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Abstract

Mice that lacked manganese‑superoxide dismutase (Mn‑SOD) activity exhibited the typical pathology of dilated cardiomyopathy (DCM). The aim of the present study was to investigate the effect of supplementation with omega‑3 polyunsaturated fatty acids (n‑3 PUFA) on heart function and oxidative stress biomarkers in mice with DCM. In the present study, heart/muscle‑specific Mn‑SOD‑deficient mice (H/M‑Sod2‑/‑) were treated with n‑3 PUFA (30 mg/kg/day) for 10 weeks, and the reactive oxygen species (ROS) production in their heart mitochondria and cardiac function was subsequently assessed. n‑3 PUFA treatment diminished ROS production and suppressed the progression of cardiac dysfunction. Furthermore, n‑3 PUFA treatment effectively reversed the cardiac dysfunction and dilatation observed in symptomatic H/M‑Sod2‑/‑ mice. Notably, n‑3 PUFA treatment ameliorated a molecular defect in connexin 43. Hematoxylin‑eosin staining indicated that the phenotype of DCM was also ameliorated following n‑3 PUFA treatment. Furthermore, echocardiography demonstrated that cardiac function was significantly improved in the mice treated with n‑3 PUFA (P<0.05). Meanwhile, pre‑treatment with n‑3 PUFA significantly decreased cardiomyocyte apoptosis (P<0.001). In conclusion, n‑3 PUFA treatment is able to prevent murine DCM, primarily by reducing ROS production and improving myocardial apoptosis. Therefore, the impairment of ROS production is proposed as a potential therapy for DCM.

Introduction

As a progressive heart muscle disease, dilated
cardiomyopathy (DCM) is characterized by a dilated ventricular
chamber, including impaired contraction of the left or both
ventricles (1,2). DCM typically leads to congestive heart
failure, thereby enhancing morbidity and mortality among patients
(3,4). Several clinical studies have indicated
that reactive oxygen species (ROS) are important in the
pathogenesis of cardiovascular diseases, including DCM (5–7).
Furthermore, it has been suggested that an imbalance between ROS
production and antioxidant defenses may result in oxidative
stress-related disorders, including DCM (8,9).
Epidemiological studies show that patients with cardiovascular
diseases can be prevented from ROS injury through antioxidant
reagents (10). Additionally,
several potential sources of ROS have been proposed, including
mitochondrial respiratory chain enzymes, xanthine oxidase,
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and
nitric oxide synthase (11).
Abnormal ROS production may further result in aberrant
morphological or functional changes, which are demonstrated as
cardiac dysfunction (12).

Omega-3 polyunsaturated fatty acid (n-3 PUFA), a
free radical scavenger, has long been suggested to be effective for
combating oxidative stress (13–15).
However, the specific mechanism through which n-3 PUFA protects the
heart from DCM remains to be elucidated. Therefore, the present
study aimed to determine the potential role of n-3 PUFA on DCM.

It has previously been indicated that the typical
pathology of DCM is exhibited by mice lacking manganese-superoxide
dismutase (Mn-SOD) activity (16,17).
Therefore, the present study used such mice as a DCM model to
explore the effect of n-3 PUFA on the parameters of heart function
and oxidative stress biomarkers in DCM mice.

Materials and methods

In vivo study protocols

A total of 8 male heart/muscle-specific
Mn-SOD-deficient (H/M-Sod2−/−) mice (age, 8 weeks;
weight, 22.5±3.1 g) were purchased from The Fourth Affiliated
Hospital of Harbin Medical University (Harbin, China). The mice
were kept in a temperature-(20–24°C) and humidity-controlled
(45–55%) environment, under a 12-h light/dark cycle, with free
access to food and water. H/M-Sod2−/− mice were fed with
3 mg/kg/day n-3 PUFA (n=4) (Sigma-Aldrich; Merck KGaA, Darmstadt,
Germany) or vehicle (n=4) (lauric acid, Sigma-Aldrich; Merck KGaA)
(18) for 10 weeks, and cardiac
function was evaluated by echocardiography. Mice were subsequently
sacrificed, hearts were harvested and the proteins extracted. The
present study was approved by the Zhejiang Province Hospital of
Integrated Traditional Chinese and Western Medicine (Hangzhou,
China).

Histological studies

For the histological analysis, heart tissues were
fixed at room temperature for 10 min in 10% buffered formalin.
Fixed tissues were subsequently dehydrated, embedded in paraffin,
sectioned into 4-µm slices and stained with hematoxylin and eosin
(H&E). Furthermore, Masson's trichrome was used to stain
myocardial sections in order to evaluate the cardiomyocyte diameter
and degree of fibrosis. Images were captured with a Pixera Pro600EX
camera and a VANOX-S microscope (Olympus Corporation, Tokyo,
Japan). Additionally, the fibrotic area and cardiomyocyte diameter
(>30 cells) were quantified with Qwin Plus V3 (Leica
Microsystems, Inc., Buffalo Grove, IL, USA), and the collagen
volume percentage was calculated as the mean of 5 fields for each
animal.

Hoechst 33258 staining

Primary cardiomyocytes were cultured in 6-well
tissue culture plates (1×105 cells per well). The cells
were incubated at 37°C in serum-free DMEM for 16 h at 70–80%
confluence. The cells were then washed three times with cold PBS
and fixed with 4% formaldehyde (Zhongshan Technology Co., Ltd.,
Zhongshan, China) in PBS for 20 min at room temperature. Next, the
cells were washed three times with cold PBS and stained with
Hoechst 33258 (10 µg/ml; 50 µl/slide; Sigma-Aldrich; Merck KGaA) at
room temperature for 5 min. Following staining, cold PBS was used
to further rinse the cells, and were then examined under a
fluorescence microscope.

Apoptosis assay

In order to detect the effects of n-3 PUFA on cell
apoptosis, primary cardiomyocytes isolated from mice were washed
with cold PBS three times. Next, flow cytometry was used to
determine cell apoptosis with an Annexin-V fluorescein
isothiocyanate-propidium iodide (FITC-PI) apoptosis kit
(Invitrogen, Carlsbad, CA, USA). In summary, the cells
(1×106) were washed with 1X PBS three times and
suspended at 2–3×106 cells/ml in X1 Annexin-V binding
buffer [10 mM HEPES/NaOH, (pH 7.4), 140 mM NaCl, 2.5 mM
CaCl2]. Annexin-V FITC and PI buffer were then added to
the cells, which were then incubated at room temperature for 15 min
in the dark. The cells that did not undergo any treatment were used
as an internal control. Following incubation, the cells were
filtered using a filter screen and analyzed using a flow cytometer
within 1 h of staining. Cell apoptosis was analyzed using BD
CellQuest Pro™ Analysis Software (BD Biosciences, San
Jose, CA, USA).

Determination and quantification of
ROS

Primary cardiomyocytes isolated from the mice were
cultured on slides in a 6-well chamber at 60% confluence at 37°C.
Two days later, the slides were washed with cold PBS three times.
Additionally, the slides were treated with 5 µM dihydroethidium
(DHE; Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) in
serum-free DMEM F-12 medium (GE Healthcare Life Sciences) for 30
min at 37°C in darkness. Furthermore, the cells were fixed in 4%
paraformaldehyde for 30 min at room temperature. The slides were
then washed with cold PBS three times and mounted. Finally,
immunofluorescence images were captured by fluorescence microscopy,
and in order to quantify the intracellular ROS, the relative
fluorescence intensities were analyzed using flow cytometry in the
primary cardiomyocytes. Briefly, 1×106 cells were
centrifuged (200 × g) for 10 min at room temperature and the
supernatant was discarded. The cell pellet was resuspended in 1 ml
of PBS at room temperature. A 2 mM solution of H2DCFDA
(Invitrogen; Thermo Fisher Scientific, Inc.) was freshly prepared
in ethanol and 5 µl was added to the cell suspension (final
concentration 10 µM) and incubated at 37°C for 20 min. Cells were
centrifuged at 200 × g for 5 min at room temperature, the
supernatant was discarded, and the cell pellet was resuspended in
500 µl of PBS. Flow cytometric analysis was performed in duplicate
using a flow cytometer and acquired data were analyzed using WinMDI
v2.8 software (BD Biosciences).

Determination of protein carbonylation
and ATP content

The nuclear and mitochondrial fractions of the heart
were isolated in order to quantify cardiac protein carbonylation.
In brief, the crude nuclear fractions were isolated from tissue
homogenates at 1,000 × g for 5 min at 4°C and washed with PBS at
4°C. The mitochondrial fractions were separated as previously
described (22). The carbonylation
of mitochondrial and nuclear protein was evaluated using an Oxyblot
protein oxidation detection kit (EMD Millipore) according to the
manufacturer's instructions. Immunoreactive spots were visualized
with enhanced chemiluminescence (GE Healthcare, Buckinghamshire,
UK) and quantified with ImageJ 1.43b (National Institutes of
Health, Bethesda, MD, USA). Furthermore, the ATP content was
determined using an ATP assay kit (colorimetric/fluorometric) (cat.
no. ab83355; Abcam), according to the manufacturer's
instructions.

Statistical analysis

Data are presented as the mean ± standard error from
three independent experiments. Statistical analysis was performed
with Student's-test. P<0.05 was considered to indicate a
statistically significant difference.

Discussion

The present study identified that n-3 PUFA treatment
was able to partially abolish cardiac enlargement and dysfunction
in H/M-Sod2−/− mice and that the protective effects of
n-3 PUFA predominantly originated from reduced ROS production and
cardiomyocyte apoptosis. Together, these data indicate that
decreased oxidative damage contributes to the reduction of cardiac
enlargement in H/M-Sod2−/− mice.

Knockout of the Mn-SOD gene in the heart and muscle
may lead to cardiac oxidative stress, which leads to contractile
dysfunction, fibrosis and myocyte damage (23). According to echocardiographic
analysis, hearts from H/M-Sod2−/− mice were
significantly enlarged. Furthermore, EF and FS were also found to
be reduced in the hearts of H/M-Sod2−/− mice, indicating
severe DCM and cardiac dysfunction of these mice. Notably, n-3 PUFA
treatment was demonstrated to improve histological abnormalities in
DCM hearts of H/M-Sod2−/− mice, such as fibrosis,
compared with hearts from the control group. This observation is in
accordance with the Masson's trichrome-stained section analysis and
suggests that fibrosis is improved in DCM hearts.

ROS are widely associated with age-related diseases,
such as Alzheimer's and Parkinson's disease, and heart failure
(24,25). During mitochondrial respiration,
small amounts of mitochondrial ROS production may be cleared by
scavenging systems. In the presence of NOX4, superoxide anions were
significantly decreased in dilated cardiomyopathy, suggesting the
protective role of NOX4 on ROS production in HF (26,27). In
accordance with the above observations, n-3 PUFA treatment was
found to significantly increase the protein level of NOX4 in DCM
hearts of H/M-Sod2−/− mice, suggesting the protective
role of n-3 PUFA in DCM. These results indicate that mitochondrial
dysfunction may be improved by n-3 PUFA treatment in DCM.

It is widely reported that abnormal ROS production
and apoptosis are important in the pathology of DCM (28,29).
Therefore, to prevent diabetic cardiomyopathy, it is important to
simultaneously inhibit oxidative stress and apoptosis. The present
study explored the effects of n-3 PUFA on cardiomyocyte apoptosis.
It was observed that n-3 PUFA treatment led to a significant
reduction of cell apoptosis in primary cardiomyocytes isolated from
H/M-Sod2−/− mice compared with the untreated group.
Accordingly, the protein level of cleaved-caspase 3 was
significantly decreased following n-3 PUFA treatment. Furthermore,
it was also shown that n-3 PUFA demonstrated anti-inflammatory
effects since the phosphorylation levels of JNK and NF-κB were
significantly decreased.

In conclusion, oxidative stress was shown to
increase in the DCM model of H/M-Sod2−/− mice. Notably,
the results indicate that n-3 PUFA may be used as an antioxidant to
protect hearts from DCM, primarily by reducing ROS production and
cardiomyocyte apoptosis. Finally, the present study may assist in
the development of a novel therapy and prevention for DCM in human
patients.